CircRNA_100395通过结合miR-144-3p抑制心肌成纤维细胞中纤维化相关基因的表达

温艺红; 杨真祯; 朱杰宁; 符永恒; 方咸宏; 单志新

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CircRNA_100395通过结合miR-144-3p抑制心肌成纤维细胞中纤维化相关基因的表达

更新时间：2023-11-06

- CircRNA_100395通过结合miR-144-3p抑制心肌成纤维细胞中纤维化相关基因的表达
- CircRNA_100395 Inhibits Expression of Myocardial Fibrosis-related Genes in Human Atrial Myofibroblasts via Sponging miR-144-3p
- 中山大学学报（医学科学版） 2020年41卷第6期
- 作者机构：
  
  1. 华南理工大学医学院,广东,广州,510006
  2. 广东省临床药理学重点实验室，广东省人民医院//广东省医学科学院,广东,广州,510080
  3. 华南理工大学生物科学与工程学院,广东,广州,510006
  4. 南方医科大学第二临床医学院,广东,广州,510280
- 作者简介：
- 基金信息：
  
  国家自然科学基金（81770264）;广东省医学科研基金（B2020215）;广州市科技计划项目（202002030013）
- DOI：
  中图分类号： R363
- 网络首发：2020-11-20，
  
  纸质出版：2020
- 稿件说明：
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温艺红, 杨真祯, 朱杰宁, 等. CircRNA_100395通过结合miR-144-3p抑制心肌成纤维细胞中纤维化相关基因的表达[J]. 中山大学学报（医学科学版）, 2020,41(6).

CircRNA_100395 Inhibits Expression of Myocardial Fibrosis-related Genes in Human Atrial Myofibroblasts via Sponging miR-144-3p[J]. Journal of Sun Yat-sen University (Medical Sciences), 2020, 41(6).
温艺红, 杨真祯, 朱杰宁, 等. CircRNA_100395通过结合miR-144-3p抑制心肌成纤维细胞中纤维化相关基因的表达[J]. 中山大学学报（医学科学版）, 2020,41(6). DOI：

CircRNA_100395 Inhibits Expression of Myocardial Fibrosis-related Genes in Human Atrial Myofibroblasts via Sponging miR-144-3p[J]. Journal of Sun Yat-sen University (Medical Sciences), 2020, 41(6). DOI：

摘要

【目的】研究环形 RNA circRNA_100395 调节心肌成纤维细胞中纤维化相关基因表达的作用机制。【方法】CircRNAs 表达谱芯片分析结合实时荧光定量 PCR 验证 circRNA_100395 在长期持续性房颤（AF）病人左心耳组织中的表达。检测血管紧张素Ⅱ（Ang-Ⅱ）诱导人心房肌成纤维细胞（HAFs）中circRNA_100395 的表达及在细胞核质中分布情况。利用放线菌素D 实验检测circRNA_100395 的RNA 稳定性。制备重组circRNA_100395 腺病毒（rAd-circRNA_100395）并感染 HAFs，检测 HAFs 中纤维化相关基因 Col1a1、Col3a1 和 Acta2 的 mRNA 和蛋白表达。通过双荧光素酶报告基因实验和RNA pull-down 实验分别鉴定circRNA_100395 与微小RNA miR-144-3p 的结合作用。【结果】Masson 染色结果显示 AF 病人心耳组织的纤维化明显加重（P < 0.01）。CircRNA_100395 在AF 患者心耳组织中表达降低（P < 0.05），但在Ang-Ⅱ诱导的HAFs 中表达明显升高（P < 0.05），且主要分布于HAFs 细胞质内。放线菌素D 实验证明 circRNA_100395 的 RNA 稳定性明显高于其宿主基因 KLHL20 mRNA。过表达circRNA_100395 抑制 HAFs 中纤维化相关基因表达。双荧光素酶报告基因实验和 RNA Pull-down 实验均证实circRNA_100395 与miR-144-3p 存在特异性的结合作用。CircRNA_100395 可抑制miR-144-3p 促HAFs 中纤维化相关基因表达的作用。【结论】CircRNA_100395 通过特异结合miR-144-3p 来发挥抑制纤维化相关基因表达的作用。

Abstract

【Objective】To investigate the effect of circRNA_100395 on myocardial fibrosis and explore its mechanism.【Methods】Circular RNA（circRNA）microarray was performed to show the differential expression of circRNAs in the left atrial appendage between patients with long-standing persistent atrial fibrillation（AF）and the healthy controls， and the expression of circRNA_100395 was detected by RT-qPCR，as well as its distribution in the cytoplasm and nucleus of human atrial fibroblasts（HAFs）. Expression of circRNA_100395 and its host gene KLHL20 was detected in angiotensin Ⅱ（Ang-Ⅱ）-induced HAFs by RT-qPCR assay. Actinomycin D treatment was performed to test the RNA stability of circRNA_100395 in HAFs. Over-expression of circRNA_100395 was achieved in HAFs with infection of the recombinant circRNA_100395 adenovirus （rAd- circRNA_100395）. The expression of Col1a1，Col3a1 and Acta2 was detected in HAFs by RT-qPCR and Western blot assay，respectively. Dual luciferase reporter assay and RNA pull-down assay were performed to identify the interaction between circRNA_100395 and miR- 144- 3p. 【Results】Masson staining revealed that myocardial fibrosis was markedly increased in the left atrial appendage of AF patients（P < 0.01）. CircRNA_100395 was decreased in the left atrial appendage of AF patients（P < 0.05），but was increased in Ang-II-induced HAFs（P < 0.05）， with high enrichment in the cytoplasm of HAFs. CircRNA_100395 was more stable than mRNA of its host gene of KLHL20 when it was subjected to actinomycin D treatment. Overexpression of circRNA_100395 significantly inhibited the expression of fibrosis- related genes in HAFs. Bioinformatics predicted the potential binding site of miR- 144-3p in circRNA_100395 ，and the specific interaction between circRNA_100395 and miR-144-3p was confirmed by the dual luciferase reporter assay and RNA pull-down assay ，respectively. Overexpression of miR- 144-3p contributed to the expression of fibrosis-related genes in HAFs，while circRNA_100395 could abolish the pro-fibrosis effect of miR- 144- 3p.【Conclusion】CircRNA_100395 exhibits an anti-fibrosis effect in HAFs through sponging miR-144-3p.

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